Mutagenesis, Vol. 15, No. 3, 229-234,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity
1 Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, R. da Junqueira 96, P-1349-008 Lisbon, Portugal, 2 Faculty of Sciences and Technology, New University of Lisbon, Lisbon, Portugal, 3 Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal and 4 University Lusófona, Lisbon, Portugal
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Abstract |
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Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and Byssochlamys. Patulin is a common contaminant of ripe apples used for the production of apple juice concentrates and is also present in other fruits, vegetables and food products. Patulin has been reported to have mutagenic, carcinogenic and teratogenic properties. Nevertheless, these properties are still a matter of debate. In this report, we further investigated the genotoxicity of patulin in mammalian cells by two different approaches. Firstly, we evaluated the induction of micronuclei in cytokinesis-blocked human lymphocytes. This approach is important because available data concerning the genetic toxicity of patulin in human cells is sparse. Secondly, we chose an established model for patulin genotoxicity, i.e. the chromosomal aberration assay in V79 Chinese hamster cells, to clarify whether concomitant exposure to ascorbic acid with the mycotoxin modulates or not the clastogenicity of patulin. The results unequivocally show induction of DNA-damaged cells by patulin as assessed by both cytogenetic assays. In addition, an almost complete abolition of patulin (0.8 µM) clastogenicity was observed in the presence of 80 µM ascorbic acid (P < 0.05), showing that although a genetic risk is present, ascorbic acid could somehow partially modulate this problem.
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Introduction |
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Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one) (Figure 1
) is a mycotoxin produced by several species of Penicillium, Aspergillus and Byssochlamys, with the most commonly encountered species being Penicillium expansum (IARC, 1986). It is a common contaminant of ripe apples used for the production of apple juice concentrates and it has been shown to be mutagenic (Korte, 1980; Thust et al., 1982; Roll et al., 1990; Pfeiffer et al., 1998), carcinogenic (Dickens and Jones, 1961) and teratogenic (Roll et al., 1990). It also has been detected in other fruits such as pears, apricots, peaches and grapes. This compound is, however, classified by the IARC in Group 3, since this agency considers the evidence for carcinogenicity in experimental animals inadequate. With respect to human data, no case reports or epidemiological studies of the carcinogenicity of patulin are available (IARC, 1986). Recently Llewellyn et al. (1998) evaluated the immunotoxicity of patulin but the data obtained suggest that it is not toxic to the immune system in female B6C3F1 mice. However, concerns about the toxic properties of patulin have led various countries and the World Health Organization to establish 50µg/l as the recommended limit in apple juice (van Egmond, 1987).
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The mutagenicity of patulin is also a matter of discussion. This compound did not increase revertant frequency in the Ames test using several strains (reviewed in IARC, 1986; Würgler et al., 1991). This is the typical behaviour of patulin in gene mutation assays, but some reports have shown mutagenic activity in Saccharomyces cerevisae strains and in Bacillus subtilis (reviewed in IARC, 1986).
Patulin induced chromatid-type aberrations in V79-E Chinese hamster cells only in the absence of S9 mix from the culture medium (Thust et al., 1982). Patulin also induced micronuclei in V79 Chinese hamster cells, suggesting both aneugenic and clastogenic properties (Pfeiffer et al., 1998), but did not increase sister chromatid exchange frequency in this cell line (Thust et al., 1982). In vivo cytogenetic studies suggested the induction of chromosome damage in Chinese hamster bone marrow cells (Korte, 1980; Roll et al., 1990).
The cellular mechanism of patulin toxicity is attributed to binding to sulphydryl groups in proteins and amino acids in the plasma membrane, which is supported by a reduction in glutathione levels and subsequent oxidative damage (Riley and Showker, 1991).
In this report, we further investigate the genotoxicity of patulin in mammalian cells using two different approaches. (i) Evaluation of the induction of micronuclei in cytokinesis-blocked human lymphocytes. This issue is important since available data concerning the genetic toxicity of patulin in human cells is sparse. In a review by the IARC, an increase in sister chromatid exchange in human lymphocytes was reported, as well as an increase in chromosomal aberrations in human peripheral leukocytes, but no increase in unscheduled DNA synthesis was observed in cultured human embryonic liver cells (IARC, 1986). (ii) The chromosomal aberrations assay in V79 Chinese hamster cells, an established model to study the genotoxicity of patulin. This was to clarify an important question, whether concomitant exposure to ascorbic acid with the mycotoxin can modulate the clastogenicity of patulin, since several reports have shown that ascorbic acid can decrease the stability of patulin in aqueous solutions (Brackett and Marth, 1979; Doores, 1983; Fremy et al., 1995).
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Materials and methods |
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Chemicals and culture media
Patulin, ascorbic acid, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), mitomycin C (MMC), newborn and foetal calf serum and Ham's F-10 medium, cytochalasin B, L-glutamine, penicillin and streptomycin were purchased from Sigma (St Louis, MO). Dimethylsulphoxide (DMSO), methanol, acetic acid, potassium chloride and Giemsa dye were obtained from Merck (Darmstadt, Germany). A mixture of penicillin, streptomycin and amphotericin B was from Irvine Scientific (Santa Ana, CA). Colchicine was purchased from Fluka (Buchs, Switzerland). Trypsin was obtained from Difco Laboratories (Detroit, MI). Phytohaemagglutinin (PHA) (HA 15) was obtained from Murex (Dartford, UK) and reconstituted in 5 ml of sterile water. Heparin was purchased from Braun (Melsungen, Germany).
Cytokinesis-blocked human lymphocyte micronuclei
Aliquots of 500 µl of whole blood from seven healthy donors were cultured in 4.5 ml Ham's F-10 medium supplemented with 24% foetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), 1% L-glutamine and 1% heparin (50 IU/ml). Lymphocytes were stimulated using 25 µl of PHA and incubated at 37°C. At 24 h culture the cells were exposed to different doses of patulin (2.5–7.5 µM) dissolved in DMSO. These doses were chosen from the range of non-cytotoxic concentrations as assessed by the cytokinesis-blocked proliferating index (CBPI). This index was calculated according to the following formula: 
as proposed by Surrallés et al. (1995), where MI–MIV represent the number of human lymphocytes with one to four nuclei and n the total number of cells scored.
Cytochalasin B was added after 44 h at a final concentration of 6 µg/ml. After a total of 72 h culture, cells were harvested by centrifugation, treated twice with 5 ml of a mixture (pH 7.2) of RPMI 1640 and deionised water 4:1, supplemented with 2% foetal calf serum. The cells were again centrifuged and submitted to a mild hypotonic treatment in a mixture (pH 7.2) of RPMI 1640 and deionised water 1:4, supplemented with 2% foetal calf serum, and immediately centrifuged. The centrifuged cells were placed on dry slides and smears were made. After air drying the slides were fixed with freshly prepared cold methanol/acetic acid (3:1) for 20 min. One day later the slides were stained with 4% Giemsa in 0.01 M phosphate buffer, pH 6.8, for 8 min.
For each experiment, 1000 binucleated lymphocytes with well preserved cytoplasm were scored. Micronuclei (MN) were identified according to the criteria of Caria et al. (1995) using a 500x magnification for detection and a 1250x magnification for confirmation. Control cells included a DMSO control, which did not exceed 0.3% (v/v) of the culture medium, and MMC as a positive control (0.75 µM).
Statistical analysis for the comparison of each test dose to the DMSO control was performed using Student's two-tailed t-test. In order to simultaneously compare all the test doses in the dose–response curve, a non-parametric ANOVA (Friedman test) was carried out.
MTT cytotoxicity assay
Wild-type V79 Chinese hamster cells (MZ) were kindly provided by Prof. H.R. Glatt (Mainz and Postdam, Germany).
The MTT assay (Mitchell, 1988) was performed as follows. Approximately 0.5x104 V79 cells were grown for 24 h in 12-well plates (Corning Costar, Corning, NY) in 1 ml Ham's F-10 medium supplemented with 10% newborn calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin B (0.25 µg/ml) and incubated at 37°C under an atmosphere containing 5% carbon dioxide. Different doses of patulin (0.35–1.55 µM) were then added and the cells were incubated for 20 h. The medium was then removed, the cells were washed with phosphate-buffered saline and then MTT (0.5 mg/ml dissolved in culture medium) was added for a further 4 h. Cells were treated with DMSO and the absorbance of the converted dye was measured at 570 nm. Cytotoxicity was assessed by comparing the absorbance values of treated cells with control cells. Triplicate assays were performed.
Chromosomal aberration assay
V79 Chinese hamster cells were cultured as described above in 5 ml Ham's F-10 medium supplemented with 10% newborn calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin B (0.25 µg/ml) and incubated at 37°C under an atmosphere containing 5% carbon dioxide.
In the first set of experiments to evaluate the genotoxicity of patulin, 24 h cultures (~5x105 cells) growing as monolayers in 25 cm3 tissue culture flasks (Greiner, Frickenhausen, Germany) were treated with different doses of patulin (0.35–0.95 µM) dissolved in DMSO. These doses were chosen from the range of non-cytotoxic doses as assessed by the MTT assay, as described above, and by the mitotic index (MI).
In the set of experiments to test the anticlastogenic effect of ascorbic acid on the genotoxicity of patulin, three doses of ascorbic acid (0.8, 8.0 and 80.0 µM) were incubated together with a genotoxic dose of patulin (0.80 µM). In all the experiments, the cells were grown for an additional period of 17 h and afterwards the medium was removed and the cells were washed. Colchicine was added at a final concentration of 0.6 µg/ml. Cells were grown for a further 3 h and then harvested by trypsinisation.
After 3 min hypotonic treatment with 75 mM KCl at 37°C, cells were fixed with methanol/acetic acid (3:1) and slides were prepared and stained with Giemsa (4% v/v in 0.01 M phosphate buffer, pH 6.8) for 10 min. Three or four independent experiments were carried out for each dose and 100 metaphases were scored. Control cells included a DMSO control, which did not exceed 0.5% (v/v) of the culture medium, and MMC as positive control (0.75 µM). Scoring of the different types of aberrations followed the criteria described by Rueff et al. (1993).
The statistical analysis for comparison of each test dose of patulin to the DMSO control as well as for comparison of ascorbic acid anticlastogenic effects on patulin genotoxicity was performed using Student's two-tailed t-test. In order to simultaneously compare all the test doses in the dose–response curve, a non-parametric ANOVA (Friedman test) was also carried out.
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Results |
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Table I
presents data concerning the micronucleus assay in cytokinesis-blocked human lymphocytes from seven healthy donors. In Table I, besides the frequency of micronucleated cytokinesis-blocked lymphocytes, the frequency of binucleated lymphocytes [BN (%)] as well as the proliferating index CBPI are presented as measures of cytotoxicity. Figure 2 shows the average MNBN values (
) and respective standard deviations from the seven donors studied. The micronucleated cells increased significantly and in a dose–response manner for the three concentrations studied, 2.5, 5.0 and 7.5 µM (P < 0.05, P < 0.001 and P < 0.001, respectively). The non-parametric ANOVA for simultaneous comparison of all the doses tested revealed a P value <0.001. The highest concentration tested augmented yields of DNA-damaged cells ~3.6-fold when compared with background levels. Cytotoxicity, as assessed by the mentioned parameters [BN (%) and CBPI], was not present at the concentrations studied. A clear induction of micronuclei in human cells by patulin is shown in both Table I and Figure 2.
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Table II
presents the raw data from three independent experiments performed with five concentrations of patulin with V79 Chinese hamster cells in the absence of any metabolic activation. In Table II the different individual aberrations, the frequencies (%) of cells with chromosomal aberrations including (CAIG) and excluding gaps (CAEG) as well as the MI are presented. Under these experimental conditions, patulin induces not only chromatid and isochromatid aberrations, which are more frequent, but also some chromosomal aberrations in V79 cells. Clastogenic activity was detectable in a very narrow dose range up to concentrations of 1 µM. For higher concentrations patulin is a potent toxic agent for V79 cells (MZ). The toxicity of patulin to V79 cells, as assessed by the MTT test, is presented in Figure 3. As shown, for concentrations >1 µM there is a considerable decrease in cell viability. Figure 4 presents the average frequencies of CAEG (%) and respective standard deviations from the above- mentioned independent experiments. CAEG increased in a dose-dependent manner up to 0.8 µM (~10-fold the background genotoxicity to DMSO control cells). The genotoxicities of the doses 0.65, 0.8 and 0.95 µM were significantly different from that observed in DMSO control cells (P < 0.05, P < 0.05 and P < 0.01, respectively). Non-parametric ANOVA for simultaneous comparison of all the doses tested revealed a P value <0.05. Figure 4 and Table II unequivocally show genotoxicity of patulin in V79 cells.
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Table III
presents the raw data from four independent experiments with V79 Chinese hamster cells performed with three different concentrations of ascorbic acid (0.8, 8.0 and 80.0 µM) simultaneously incubated with the highest genotoxic dose of patulin (0.8 µM) tested in the previous experiments. These experiments were performed under the same conditions as the former set of experiments, using the chromosomal aberration assay as the end-point. Figure 5 represents the pooled data for CAEG from the above-mentioned experiments (average values ± SD). A decrease in the clastogenicity of patulin in the presence of ascorbic acid was observed. However, ascorbic acid was only completely efficient as an anticlastogenic agent at the dose of 80 µM, significantly reducing patulin clastogenicity. It should be noted that the baseline concentration of ascorbic acid in culture is essentially due to the serum used, since Ham's F-10 medium does not contain ascorbic acid. The statistical analysis revealed that CAEG from DMSO control cultures and patulin plus 80 µM ascorbic acid cultures did not differ significantly.
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Discussion |
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Patulin is a toxic contaminant present at significant concentrations in some food products. There is particular concern regarding apples and apple products (e.g. concentrated juice), but pears, baby food, mouldy beetroot, mouldy bread and other fruit and fruit juices also have appreciable concentrations of this contaminant (reviewed in IARC, 1986; Paster et al., 1995). Recently Gökmen and Acar (1998) found patulin in apple juice at concentrations varying from 7 to 376 µg/l. This mycotoxin has been described as a clastogen and recently also as an aneugen (Pfeiffer et al., 1998
) in mammalian cells. Almost all the evidence for clastogenic activity of patulin either in vitro or in vivo was reported for Chinese hamster cells. Very few reports have focussed on this effect in human cells, namely in stimulated peripheral blood lymphocytes. This report addresses this issue (Table I
and Figure 2), using the cytokinesis-blocked micronucleus assay in human lymphocytes. The inherent advantages of this assay have been described elsewhere (Fenech, 1993). The three doses chosen revealed an increase in a dose-dependent manner in cytokinesis-blocked micronucleated lymphocytes. The doses studied were not cytotoxic and interestingly both the cytotoxic and genotoxic potential of patulin in human lymphocytes seemed to be less evident than in V79 cells. In fact, if we compare both types of cells, regardless of the differences in these end-points, the yields of DNA-damaged V79 cells even with doses ~10-fold lower are markedly higher than those in human lymphocytes. Erythrocytes have a variety of enzymatic activities which can detoxify numerous toxic agents and the presence of erythrocytes in whole blood cultures of lymphocytes may help to explain the lower cytotoxicity and genotoxicity of patulin (for a review see Rueff et al., 1996). On the other hand, one cannot rule out the possibility of patulin reacting with erythrocyte targets and thus lowering the effective concentration of the mycotoxin. Additionally, the differences observed could also be due to the end-point chosen.
The results presented in Table II and Figure 4 show the clastogenic potential of patulin in V79 cells at very low doses. This compound is rather cytotoxic at doses higher than 1 µM, as revealed by a substantial reduction in MI and by means of a widely used survival test, the non-clonogenic MTT assay (Figure 3). The concentration of 0.95 µM patulin seems to be less genotoxic than 0.8 µM, which could be explained by an increase in cytotoxicity.
The results obtained in V79 Chinese hamster cells confirm published results (Thust et al., 1982) concerning the existence of genotoxic potential and the pattern of genotoxicity presented. Under our experimental conditions, however, genotoxicity was present at even lower concentrations (up to 10-fold) of patulin when compared with the results of Thust et al. (1982). These differences could be a consequence of either differences in the experimental protocol, in the clone of V79 used (Kirkland, 1992) or in the purity of patulin used, as well as cytoxicity of this compound, which was more evident in our experiments.
In this work the anticlastogenic properties of ascorbic acid towards patulin were also studied. This point is of major interest, since it is well known that apples and apple products usually contain ascorbic acid, either naturally or added as a preservative. Interestingly, patulin has not been found in citrus products which have a high content of ascorbic acid. In addition, ascorbic acid is an important factor in human antioxidant defence mechanisms. For these reasons the use of a well-studied cell system for the genotoxic assessment of patulin, V79 Chinese hamster cells, seemed to be a good choice. Moreover, this experimental design could be considered more accurate in comparison with human lymphocytes because cultured whole blood has a variable content of ascorbic acid among donors. We have also chosen this system because previous experiments (Figures 2 and 4) showed that the genotoxiciy of patulin is more evident in the chromosomal aberration assay in V79 cells than in the micronucleus assay in human lymphocytes, thus allowing easier detection of any protective effect of ascorbic acid. The selected doses of ascorbic acid decreased the genotoxicity of patulin. This was evident for the highest concentration studied, 80 µM, which substantially reduced the genotoxicity of patulin such that statistical analysis of the comparison with DMSO control cells (negative control) showed no significant difference (Figure 5). Additionally, this concentration is much lower than that present in some commercial juices (up to 1700 µM; personal communication) and is within the usual range of ascorbic acid levels (50–200 µM) found in plasma (Halliwell and Gutteridge, 1995).
The mechanism by which the genotoxicity of patulin is almost completely abolished in the presence of ascorbic acid is not yet fully understood. However, it is possible that metal-catalysed oxidation of ascorbic acid occurs. This reaction would yield singlet oxygen and a free radical form of a metal–ascorbate complex (Brackett and Marth, 1979). These chemically reactive forms could then attack the conjugated double bond of patulin, changing the molecular structure, which could account for the decrease in genotoxicity. Thus, the addition of ascorbic acid to apple juice which contains patulin might be of value, particularly in that highly contaminated with this mycotoxin. This study shows that although a genetic risk due to patulin is present, ascorbic acid can somehow partially modulate the hazards due to the presence of this mycotoxin.
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Acknowledgments |
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We are grateful to Dr L.Goncialves for invaluable assistance with the statistical analysis. This study was supported in part by the FCT (Project PRAXIS/PSAU/C 67-96) and the Luso American Foundation for Development (FLAD).
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Notes |
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5 To whom correspondence should be addressed: Tel: +351 21 3610290; Fax: +351 21 3622018; Email: rueff.gene{at}fcm.unl.ptorjose.rueff{at}gene.unl.mailpac.pt
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Received on October 11, 1999; accepted on December 13, 1999.
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